Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. An additional example of modulation of RNA target function by an occupancy-based mechanism is modulation of microRNA function. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.
The synthesis of 5′-substituted DNA and RNA derivatives and their incorporation into oligomeric compounds has been reported in the literature (Saha et al., J. Org. Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020). The 5′-substituted monomers have also been made as the monophosphate with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).
A genus of modified nucleosides including optional modification at a plurality of positions including the 5′-position and the 2′-position of the sugar ring and oligomeric compounds incorporating these modified nucleosides therein has been reported (see International Application Number: PCT/US94/02993, Published on Oct. 13, 1994 as WO 94/22890).
The synthesis of 5′-CH2 substituted 2′-O-protected nucleosides and their incorporation into oligomers has been previously reported (see Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al. Bioconjugate Chem. 1999, 10, 921-924).
Amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to 3′) comprises a 2′-OCH3 and a 5′-(S)—CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290).
A genus of 2′-substituted 5′-CH2 (or O) modified nucleosides and a discussion of incorporating them into oligonucleotides has been previously reported (see International Application Number: PCT/US92/01020, published on Feb. 7, 1992 as WO 92/13869).
The synthesis of modified 5′-methylene phosphonate monomers having 2′-substitution and their use to make modified antiviral dimers has been previously reported (see U.S. patent application Ser. No. 10/418,662, published on Apr. 6, 2006 as US 2006/0074035).
Various analogs of 5′-alkynylphosphonate ribonucleosides have been prepared and reported in the literature (see Meurillon et al., Tetrahedron, 2009, 65, 6039-6046 and Nucleic Acids Symposium Series, 2008, 52(1), 565-566; Lera et al., Org. Lett., 2000, 2(24), 3873-3875).
The preparation of 5′-vinylphosphonate DNA and RNA monomers and their use to make dimeric compounds for oligonucleotide synthesis have been described. Their biochemical studies have also been discussed (see Whittaker et al., Tet. Lett., 2008, 49, 6984-6987; Abbas et al., Org. Lett., 2001, 3(21), 3365-3367; Bertram et al., Biochemistry, 2002, 41, 7725-7731; Zhao et al., Tet. Lett., 1996, 37(35), 6239-6242 and Jung, et al., Bioorg. Med. Chem., 2000, 8, 2501-2509).
Various BNA's have been prepared and reported in the patent literature as well as in scientific literature, see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Wengel et al., PCT International Application WO 98-DK393 19980914; Singh et al., J. Org. Chem., 1998, 63, 10035-10039, the text of each is incorporated by reference herein, in their entirety. Examples of issued US patents and published applications include for example: U.S. Pat. Nos. 7,053,207, 6,770,748, 6,268,490 and 6,794,499 and published U.S. applications 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114 and 20030082807; the text of each is incorporated by reference herein, in their entirety.
The synthesis of various cyclohexitol nucleoside analogs has been reported in the literature, see for example: Verheggen et al., J. Med. Chem., 1995, 38, 826-835; Altmann et al., Chimia, 1996, 50, 168-176; Herdewijn et al., Bioorganic & Medicinal Chemistry Letters, 1996, 6 (13), 1457-1460; Verheggen et al., Nucleosides & Nucleotides, 1996, 15(1-3), 325-335; Ostrowski et al., J. Med. Chem., 1998, 41, 4343-4353; Allart et al., Tetrahedron., 1999, 55, 6527-6546; Wouters et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 1563-1566; Brown, et al., Drug Development Res., 2000, 49, 253-259; published PCT application: WO 93/25565; WO 02/18406; and WO 05/049582; U.S. Pat. Nos. 5,314,893; 5,607,922; and 6,455,507.
Various cyclohexitol nucleoside analogs have been described as monomers and have also been incorporated into oligomeric compounds (see for example: Published PCT application, WO 93/25565, published Dec. 23, 1993; Augustyns et al. Nucleic Acids Res., 1993, 21(20), 4670-4676; Verheggen et al., J. Med. Chem., 1993, 36, 2033-2040; Van Aerschol et al., Angew. Chem. Int. Ed. Engl., 1995, 34(12), 1338-1339; Anderson et al., Tetrahedron Letters, 1996, 37(45), 8147-8150; Herdewijn et al., Liebigs Ann., 1996, 1337-1348; De Bouvere et al., Liebigs Ann./Recueil, 1997, 1453-1461; 1513-1520; Hendrix et al., Chem. Eur. J., 1997, 3(1), 110-120; Hendrix et al., Chem. Eur. J., 1997, 3(9), 1513-1520; Hossain et al, J. Org. Chem., 1998, 63, 1574-1582; Allart et al., Chem. Eur. J., 1999, 5(8), 2424-2431; Boudou et al., Nucleic Acids Res., 1999, 27(6), 1450-1456; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 1108-1109; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 2653-2656; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 5856-5859; Pochet et al., Nucleosides & Nucleotides, 1999, 18 (4&5), 1015-1017; Vastmans et al., Collection Symposium Series, 1999, 2, 156-160; Froeyen et al., Helvetica Chimica Acta, 2000, 83, 2153-2182; Kozlov et al., Chem. Eur. J., 2000, 6(1), 151-155; Atkins et al., Parmazie, 2000, 55(8), 615-617; Lescrinier et al., Chemistry & Biology, 2000, 7, 719-731; Lescrinier et al., Helvetica Chimica Acta, 2000, 83, 1291-1310; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; US Patent Application US 2004/0033967; Published US Patent Application US 2008/0038745; Published and Issued U.S. Pat. No. 7,276,592). DNA analogs have also been reviewed in an article (see: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854) which included a general discussion of cyclohexitol nucleoside analogs (under the name: hexitol nucleic acid family).
Oligomeric compounds having phosphodiester linked hexitol nucleic acids (HNA, or 1,5-anhydrohexitol nucleic acids) have also been prepared for evaluation in cell assays. The different motifs that have been evaluated are fully modified wherein each monomer is a phosphodiester linked hexitol nucleic acid analog and gapped wherein each monomer in the 3′ and 5′ external regions of the oligomeric compound are each phosphodiester linked hexitol nucleic acid analogs and each monomer in the internal region is a phosphorothioate linked deoxyribonucleoside (see: Kang et al., Nucleic Acids Research, 2004, 32(14), 4411-4419; Vandermeeren et al., 2000, 55, 655-663; Flores et al., Parasitol Res., 1999, 85, 864-866; and Hendrix et al., Chem. Eur. J, 1997, 3(9), 1513-1520).
Oligomeric compounds having phosphodiester linked analogs having the 3′-OH group which are referred to in the art as ANA or D-altritol nucleic acids have been prepared and evaluated both structurally and in vitro (Allart et al., Chem. Eur. J., 1999, 5(8), 2424-2431).
Chemically modified siRNA's having incorporated hexitol nucleotides (also referred to in the art as HNA nucleic acids) have been prepared and tested for silencing capacity (see: Published PCT application, WO 06/047842, published May 11, 2006.
Cyclohexenyl nucleic acids and analogs thereof have been reported in the scientific and patent literature as monomers as well as in oligomeric compounds, see for example: Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvith et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety.
The synthesis of various tetrahydropyran nucleoside analogs has been reported in the literature, see for example: Verheggen et al., J. Med. Chem., 1995, 38, 826-835; Altmann et al., Chimia, 1996, 50, 168-176; Herdewijn et al., Bioorganic & Medicinal Chemistry Letters, 1996, 6 (13), 1457-1460; Verheggen et al., Nucleosides & Nucleotides, 1996, 15(1-3), 325-335; Ostrowski et al., J. Med. Chem., 1998, 41, 4343-4353; Allart et al., Tetrahedron., 1999, 55, 6527-6546; Wouters et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 1563-1566; Brown, et al., Drug Development Res., 2000, 49, 253-259; published PCT application: WO 93/25565; WO 02/18406; and WO 05/049582; U.S. Pat. Nos. 5,314,893; 5,607,922; and 6,455,507.
Various tetrahydropyran nucleoside analogs have been described as monomers and have also been incorporated into oligomeric compounds (see for example: Published PCT application, WO 93/25565, published Dec. 23, 1993; Augustyns et al. Nucleic Acids Res., 1993, 21(20), 4670-4676; Verheggen et al., J. Med. Chem., 1993, 36, 2033-2040; Van Aerschol et al., Angew. Chem. Int. Ed. Engl., 1995, 34(12), 1338-1339; Anderson et al., Tetrahedron Letters, 1996, 37(45), 8147-8150; Herdewijn et al., Liebigs Ann., 1996, 1337-1348; De Bouvere et al., Liebigs Ann./Recueil, 1997, 1453-1461; 1513-1520; Hendrix et al., Chem. Eur. J., 1997, 3(1), 110-120; Hendrix et al., Chem. Eur. J., 1997, 3(9), 1513-1520; Hossain et al, J. Org. Chem., 1998, 63, 1574-1582; Allart et al., Chem. Eur. J., 1999, 5(8), 2424-2431; Boudou et al., Nucleic Acids Res., 1999, 27(6), 1450-1456; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 1108-1109; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 2653-2656; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 5856-5859; Pochet et al., Nucleosides & Nucleotides, 1999, 18 (4&5), 1015-1017; Vastmans et al., Collection Symposium Series, 1999, 2, 156-160; Froeyen et al., Helvetica Chimica Acta, 2000, 83, 2153-2182; Kozlov et al., Chem. Eur. J., 2000, 6(1), 151-155; Atkins et al., Parmazie, 2000, 55(8), 615-617; Lescrinier et al., Chemistry & Biology, 2000, 7, 719-731; Lescrinier et al., Helvetica Chimica Acta, 2000, 83, 1291-1310; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; US Patent Application US 2004/0033967; Published US Patent Application US 2008/0038745; Published and Issued U.S. Pat. No. 7,276,592). DNA analogs have also been reviewed in an article (see: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854) which included a general discussion of tetrahydropyran nucleoside analogs (under the name: hexitol nucleic acid family).
Oligomeric compounds having phosphodiester linked 3′-H tetrahydropyran nucleoside analogs (also referred to in the art as HNA—hexitol nucleic acids or 1,5-anhydrohexitol nucleic acids) have been prepared for evaluation in cell assays. The different motifs that have been evaluated are fully modified wherein each monomer is a phosphodiester linked 3′-H tetrahydropyran nucleoside analog and gapped wherein each monomer in the 3′ and 5′ external regions of the oligomeric compound are each phosphodiester linked 3′-H tetrahydropyran nucleoside analogs and each monomer in the internal region is a phosphorothioate linked deoxyribonucleoside (see: Kang et al., Nucleic Acids Research, 2004, 32(14), 4411-4419; Vandermeeren et al., 2000, 55, 655-663; Flores et al., Parasitol Res., 1999, 85, 864-866; and Hendrix et al., Chem. Eur. J, 1997, 3(9), 1513-1520).
Oligomeric compounds having phosphodiester linked 3′-OH tetrahydropyran nucleoside analogs (also referred to in the art as ANA or D-altritol nucleic acids) have been prepared and evaluated both structurally and in vitro (Allart et al., Chem. Eur. J., 1999, 5(8), 2424-2431).
Chemically modified siRNA's having incorporated hexitol nucleotides (also referred to in the art as HNA nucleic acids) have been prepared and tested for silencing capacity (see: Published PCT application, WO 06/047842, published May 11, 2006.